Imaging of RNA in situ hybridization by atomic force microscopy

In this study we investigated the possibility of imaging internal cellular molecules after cytochemical detection with atomic force microscopy (AFM). To this end, rat 9G and HeLa cells were hybridized with haptenized probes for 28S ribosomal RNA, human elongation factor mRNA and cytomegalovirus immediate early antigen mRNA. The haptenized hybrids were subsequently detected with a peroxidase-labelled antibody and visualized with 3,3'-diaminobenzidine (DAB). The influence of various scanning conditions on cell morphology and visibility of the signal was investigated. In order to determine the influence of ethanol dehydration on cellular structure and visibility of the DAB precipitate, cells were kept in phosphate-buffered saline (PBS) and scanned under fluid after DAB development or dehydrated and subsequently scanned dry or submerged in PBS. Direct information on the increase in height of cellular structures because of internally precipitated DAB and the height of mock-hybridized cells was available. Results show that internal DAB precipitate can be detected by AFM, with the highest sensitivity in the case of dry cells. Although a relatively large amount of DAB had to be precipitated inside the cell before it was visible by AFM, the resolution of AFM for imaging of RNA–in situ hybridization signals was slightly better than that of conventional optical microscopy. Furthermore, it is concluded that dehydration of the cells has irreversible effects on cellular structure. Therefore, scanning under fluid of previously dehydrated samples cannot be considered as a good representation of the situation before dehydration.\ud \u

Chromatin Dynamics of the mouse β-globin locus

Lately it has become more clear that (subtle) changes in 3D organization of chromatin can either trigger transcription or silence genes or gene clusters. It has also been postulated that due to changes in chromatin structure, a change in chromatin accessibility of transcription factors (TF) to TF binding sites also becomes an important factor to the gene’s activation status. Both such changes have been ascribed to the mouse ?- haemoglobin gene cluster as a trigger to activate globin expression in the erythroid cell lineage. Early models speculated a scanning, random activation or a looping mechanism to activate globin transcription. The chromatin conformation capture (3C) technique has shown that there is a molecular interaction between various DNAse I hypersensitive (HS) sites that are located up- and downstream of the ?-globin gene cluster, the HS sites of the Locus Control Region (LCR) and the promoter by means of a dynamic looping mechanism. The clustering of the HS sites of the LCR and the up- and down- stream HS sites results in the formation of a so called Active Chromatin Hub (ACH) which is depending on at least two erythoid TF: EKLF and GATA-1. The long range interactions between the outlying HS -84/-85, -62/-60 and the 3’HS1 are depending on the presence of CTCF, a TF that is thought to play an important role in long range chromatin interactions across the whole genome. Prior to gene activation, cells of the early erythroid lineage (progenitors) already show a presence of an ACH, which is not found in non-erythroid cells. The final chromatin 3D structure consist of four major loops sizing 25- 38Kb and two minor loops within the LCR sizing 4.5 and 12Kb. To confirm this looping hypothesis (based on 3C technology) we used an in situ hybridization approach to visualize and, after image restoration, quantitatively measure the 3D conformational changes that take place within the locus in erythroid cells before and after differentiation. Globin gene activation is depending on long distance looping of the up- and downstream HS sites and the ?-major promotor to the LCR, resulting in a complex 3D chromatin structure. By staining the m?- globin loci with fluorescence labeled sequence specific probes followed by high-resolution 3D imaging and 3D volume rendering of the deconvolved images, the loci reveal changes in the geometric size and shape properties when cells are differentiated into a active globin transcribing cell. An almost 2x decrease in volume was measured, which was mostly due to a reduction of the longest length measured. This can be explained by a change in loop formation. The almost 70Kb loop between the LCR and the 3’HS1 is folded into two loops of 34 and 35Kb upon interaction of the promotor to the ACH to activate transcription. The limited decrease in volume and length when the locus was probed with an additional 5’ and 3’ end region is surprising. The 5’ end is actively participating in the looping process that stabilizes the ACH. However, the 3’ end has (until now) not been seen to be participate in ACH formation or any complex looping mechanism for globin gene activation. As this part of the locus seems to be the most un-dynamic, it could be the dominating factor that influences the fluorescent signal emitting from the probed DNA region and therefore cloud subtle changes in the chromatin folding mechanism of gene activation. Next to the dynamic chromatin folding process that is occurring between the HS-85/84 and the 3’HS1, a stretch of “rigid” DNA can prevent a DNA region containing activated genes to stay at the edge of a chromosome territory and possibly prevent a close proximity to the silencing effect of (spreading) heterochromatin. An increase in lateral and axial resolution like the 3D Structural Illumination Microscope (SIM) provides, could help solve the problem of detecting subtle 3D changes in chromatin structure. And in the near future will reveal many more details of 3D chromatin organization of not only the m?-globin locus but of many other intra-cellular processes

The 3D chromatin structure of the mouse β-haemoglobin gene cluster

Here we show a 3D DNA-FISH method to visualizes the 3D structure of the β-globin locus. Geometric size and shape measurements of the 3D rendered signals (128Kb) show that the volume of the β-globin locus decreases almost two fold upon gene activation. A decrease in length and a distinctive change in shape and surface structure of the locus are also observed. Adding 5’ and 3’end regions to the probe (175Kb) showed a less prominent change in length, shape and structure. It was shown (data not on this poster) that the physical distance between the two flanking regions shift in a similar limited manner, indicating that the flanking regions do not participate in ACH formation and thus active chromatin folding is occurring only within the locus proper

Super-resolution imaging reveals three-dimensional folding dynamics of the β-globin locus upon gene activation

The chromatin architecture is constantly changing because of cellular processes such as proliferation, differentiation and changes in the expression profile during gene activation or silencing. Unravelling the changes that occur in the chromatin structure during these processes has been a topic of interest for many years. It is known that gene activation of large gene loci is thought to occur by means of an active looping mechanism. It was also shown for the β-globin locus that the gene promoter interacts with an active chromatin hub by means of an active looping mechanism. This means that the locus changes in three-dimensional (3D) nuclear volume and chromatin shape. As a means of visualizing and measuring these dynamic changes in chromatin structure of the β-globin locus, we used a 3D DNA-FISH method in combination with 3D image acquisition to volume render fluorescent signals into 3D objects. These 3D chromatin structures were geometrically analysed, and results prior to and after gene activation were quantitatively compared. Confocal and superresolution imaging revealed that the inactive locus occurs in several different conformations. These conformations change in shape and surface structure upon cell differentiation into a more folded and rounded structure that has a substantially smaller size and volume. These physical measurements represent the first non-biochemical evidence that, upon gene activation, an actively transcribing chromatin hub is formed by means of additional chromatin looping

In vivo live imaging of RNA polymerase II transcription factories in primary cells

Transcription steps are marked by different modifications of the C-terminal domain of RNA polymerase II (RNAPII). Phosphorylation of Ser5 and Ser7 by cyclin-dependent kinase 7 (CDK7) as part of TFIIH marks initiation, whereas phosphorylation of Ser2 by CDK9 marks elongation. These processes are thought to take place in localized transcription foci in the nucleus, known as "transcription factories," but it has been argued that the observed clusters/foci are mere fixation or labeling artifacts. We show that transcription factories exist in living cells as distinct foci by live-imaging fluorescently labeled CDK9, a kinase known to associate with active RNAPII. These foci were observed in different cell types derived from CDK9-mCherry knock-in mice. We show that these foci are very stable while highly dynamic in exchanging CDK9. Chromatin immunoprecipitation (ChIP) coupled with deep sequencing (ChIP-seq) data show that the genome-wide binding sites of CDK9 and initiating RNAPII overlap on transcribed genes. Immunostaining shows that CDK9-mCherry foci colocalize with RNAPII-Ser5

Friend of Prmt1, a novel chromatin target of protein arginine methyltransferases

We describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightl